Optimization of protocol for sequencing of difficult templates.

In this paper, we have fine-tuned a DNA sequencing protocol suitable for a wide range of difficult templates. The primary goal was to evaluate a number of parameters--such as various dye terminator mixes in the presence or absence of additives, the amount of DNA or primer, and cycling protocols--about the effectiveness of reading through complex regions. We showed that the modification of a published protocol leads to significant (75%) cost reduction without forfeiting quality of the data. In the recommended protocol, we used betaine as a standard additive, but better results can be obtained when betaine and Reagent A are mixed in an equivalent ratio.